Pox viruses are very wide spread, double stranded DNA viruses that replicate in the cytoplasm of animal, bird and insect hosts. They have genomes of up to 300 kb long that encode much of the DNA replication and transcription machinery of the virus. Small pox was eradicated in 1977; however, variola virus and related viruses have recently reemerged as potential public health and bioterrorism hazards. While poxviruses have been studied for many years, a complete understanding of their life cycle has not yet been achieved. Inhibitors of DNA synthesis and RNA synthesis, mostly nucleotide analogs, exist. However, inhibitors of other stages of the viral life cycle would be very useful, and may become leads for anti-pox virus drugs. Prior to the packaging of the Viral DNA, concatemeric genomes must be cleaved into monomeric units for packaging. This event involves an inverted repeat near the end of each genome which, when extruded, forms an incompletely base-paired hairpin. This structure resembles a Holliday junction and the viral topoisomerase, which can cleave these structures, has been proposed as playing a role in the formation of the hairpin ends. Recently, a Holliday junction resolvase resembling the bacterial Holliday junction endonuclease RuvC has been identified by the Moss lab as the product of the A22R gene, and shown to be necessary for processing of late-stage viral concatemeric genomes into unit-length genomes. In the absence of A22R expression, formation of mature virus particles is delayed and greatly reduced. Using phage lambda integrase as a target, we have isolated peptides and small molecules that block resolution of Holliday junctions. In addition to blocking recombination by several tyrosine recombinases, these compounds also prevent branch migration and junction resolution by the RuvABC complex of E. coli as well as by the RecG helicase, which also branch-migrates Holliday junctions. We propose to express and purify the vaccinia virus A22R gene product (pA22R) and test whether our compounds inhibit it; we will characterize the interactions of pA22R with Holliday junctions, using both purified protein and cell extracts of vaccinia-infected cells with and without inhibitors; we will identify more Holliday junction-binding small molecules which inhibit pA22R resolution activity; we will determine the effect of the most potent molecules on the life cycle of vaccinia virus, and processing of unit-length genomes, and will test the toxicity of these compounds on eukaryotic cells.